Project Summary Repeated exposure to alcohol leads to neuroadapative changes that underlie the transition from moderate to excessive alcohol intake. Gene expression profiling studies in human alcoholics and rodents have led to the identification of a multitude of ethanol-responsive gene networks and pathways. However, there is a gap in knowledge in our understanding of how these networks are coordinated into a neuroadaptive response. One potential mechanism could involve the recruitment of transcription factors and transcriptional co-regulators that could modulate the expression of several downstream genes. However, very few studies have examined the role of transcriptional co-regulators in alcohol use disorders. Our preliminary results implicate a novel role for the transcriptional co-regulator Lim-Only 4 (LMO4) in regulating alcohol intake. LMO4 knockdown in the basolateral amygdala (BLA) led to a significant decrease in alcohol consumption in the intermittent access procedure and a significant deficit in conditioned place preference to alcohol suggesting a role for LMO4 in the BLA in regulating both alcohol consumption and reward. Unbiased transcriptome analysis of the BLA from WT and Lmo4gt/+ mice, which make 50% less Lmo4 than WT mice, using RNASeq revealed several genes that were differentially expressed including the kappa opioid receptor (Oprk1). Weighted gene co-expression network analysis (WGCNA) revealed extracellular matrix (ECM)-related genes as being dysregulated upon LMO4 knockdown. These results led us to hypothesize that a LMO4-regulated transcriptional network in the BLA regulates alcohol consumption. We propose the following specific aims to test this hypothesis. In Aim 1, we will determine whether Oprk1 functions downstream of LMO4 in the BLA to regulate alcohol consumption. We will use a combination of approaches including cell-type specific shRNA-mediated knockdown of Lmo4 and Oprk1 in BLA pyramidal neurons and chromatin immunoprecipitation to address this question. In Aim 2, we will determine whether LMO4 downregulation in the BLA causes abnormalities in the density of perineuronal nets (PNNs), a highly specialized form of ECM in the brain. We will next determine whether enzymatic dissolution of PNNs will reduce ethanol consumption. We will also determine whether a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS) 2 and sulfatase 2 (Sulf2) function downstream of LMO4 to regulate alcohol consumption. Finally, in Aim 3, we will use a viral-based translational affinity purification strategy to determine how the LMO4-regulated transcriptome changes with alcohol exposure. The proposed experiments are significant because identification of transcriptional targets functioning downstream of LMO4 to regulate alcohol consumption could lead to the identification of novel therapeutic targets to treat alcohol use disorders.